It has been reported previously that s-nitrosoglutathione (GSNO) exerts a potent vasodilatory effect in the isolated rat heart. GSNO also protected the heart against ischemia- and reperfusion-induced injury. These effects were antagonized by nitric oxide traps, thus, implicating the intermediacy of nitric oxide. However, the mechanism by which nitric oxide is released from GSNO in rat heart tissues remains unknown. It has been proposed that nitric oxide released from GSNO could be catalyzed by -glutamyl transpeptidase ( -GTP), a membrane-bound enzyme. The enzyme -GTP plays a central role in the catabolism of glutathione (GSH). It catalyzes the transfer of the -glutamyl moiety of GSH and related GSH derivatives to a number of acceptors. When the nucleophile is water, -GTP catalyzes the hydrolysis of GSH to cysteinylglycine (Gly-CySH) and glutamate. The metabolism of GSNO by -GTP should lead to S-nitroso-cysteinylglycine (Gly-CySNO) and glutamate. In the presence of metal ion chelators, Gly-CySNO will rapidly decompose to nitric oxide. The following conclusions were reached: 1) -GPT catalyzes the decomposition of GSNO to Gly-CySNO and glutamate. The KM for GSNO was calculated to be 28 fM. 2) -GTP catalyzed decomposition of GSNO is inhibited by -GTP inhibitors. This suggests that -GTP catalyzes the decomposition of both GSNO and GSH by the same mechanism. 3) In the absence of metal ion chelator (DTPA), -GTP catalyzed the release of nitric oxide from GSNO. The mechanism for this is the conversion of GSNO to Gly-CySNO, which is more susceptible to transition metal ion decomposition than GSNO. 4) Inhibitors of -GPT had no effect on the GSNO-dependent increase in coronary vasodilation. GSNO decomposition was enhanced by rat heart homogenate by a mechanism that was unaffected by -GTP inhibitors and by transition metal ion chelators. This suggests that -GTP is not responsible for GSNO metabolism to nitric oxide in the isolated rat heart perfusion model. These findings suggest that the effects of GSNO are not mediated by transition metal ions and -GTP. The mechanism by which GSNO elicits a vascular response is unknown and is under investigation.